Superoxide Production Increases in Nucleus Tractus Solitarius (NTS) Neurons in Rat Brain Slices during Acute Normobaric Hyperoxia and Hypoxia

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We previously reported that hyperbaric hyperoxia stimulates firing rate of putative CO2-chemoreceptors in the solitary complex of the dorsocaudal medulla oblongata in rat brain slices (JAP 95: 910-921, 2003). We next reported that the typical control level of 95%O2 is a greater source of redox stress than ≤ 40%O2 leading to increased cell death in brain slices (J. Neurophysiol. 98:1030-1041, 2007). In the present study we used 20-40%O2 as the control to test the hypothesis that normobaric hyperoxia and hypoxia increase the rate of superoxide production (·O2-) in NTS neurons. Brain slices (400μm, 36-37oC) were maintained using 1- or 2-sided superfusion. Brainstem neurons maintained in 20-40%O2 (5%CO2, balance N2) exhibited i) whole-cell/intracellular activity for many hours, ii) CO2 chemosensitivity (10-15%CO2) and iii) were stimulated by hyperoxia (60-95%O2). ·O2- production was measured (3 min intervals) using the fluorogenic probe, dihydroethidium (2.5μM), continuously loaded via the superfusate. The rate of ·O2- production (slope of fluorescence intensity units/min, FIU/min) increased during acute hyperoxia (20 to 95%O2, 15-20min). Likewise, FIU/min increased during hypoxia (40/20% to 0%O2, 10-20min). ·O2- production during hypoxia was dependent on a lower threshold tissue pO2 that is estimated to be well below 20 Torr based on measurements of tissue slice pO2. ·O2- production during hypoxia was repeatedly induced using 95%N2-5%CO2 during either 1) 1-sided slice superfusion or 2) in combination with an O2-scavenger (1mM Na2SO3) during 2-sided slice superfusion. Myxothiazol (10μM; an inhibitor of Complex III) decreased ·O2- production during hypoxia but had little effect during hyperoxia. This suggests that mitochondrial Complex III is the primary source of ·O2- during hypoxia but not hyperoxia in NTS neurons. Preliminary experiments in CA1 hippocampus and Inferior olive indicate that these neurons do not increase their rate of ·O2- production during hypoxia/Na2SO3. We posit that the similar pattern of ·O2- production in NTS neurons activated by hypoxia and hyperoxia renders these cardio-respiratory neurons vulnerable to redox stimulation and/or stress during sleep disordered breathing (episodic hypoxia, reoxygenation and rebound hyperoxia) and during exposure to normobaric and hyperbaric hyperoxia


Presented at the Society for Neuroscience Annual Meeting, Washington, DC, November 15-19, 2008.

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